Co-immunization with plasmids coding the full length and a soluble form of glycoprotein D of HSV-2 induces protective cellular and humoral immune response in mice

At present, the significance of antibody for protection of the female genital tract against infection with HSV-2 remains controversial.In the present study, the ability of a DNA vaccine encoding different forms of glycoprotein D (gD) of herpes simplex virus-2 (HSV-2) to induce simultaneously cellular and humoral responses was evaluated. Mice immunized with a plasmid encoding full length gD (pgD) developed a strong cellular immune response but weak antibody titers in serum and vaginal washings. On the other hand, mice immunized with a plasmid encoding soluble form of gD (pdeltagD) showed high titers of antibodies but a very weak cell-mediated immune response. When mice were immunized simultaneously with both plasmids, cellular and humoral immune responses were elicited. This mice showed neutralizing antibodies in serum and vaginal washings as well as a high number of IFN-gamma secreting cells in spleen. When challenged with 50 lethal doses of virus, mice immunized with pgD along with pdeltagD showed a more complete protection than mice immunized with pgD alone. Collectively these results suggest that neutralizing antibodies help cell-mediated immune response for the protection against HSV-2 infection.     

Induction of human cytomegalovirus (HCMV)-glycoprotein B (gB)-specific neutralizing antibody and phosphoprotein 65 (pp65)-specific cytotoxic T lymphocyte responses by naked DNA immunization

Plasmids expressing the human cytomegalovirus (HCMV) glycoprotein B (gB) (UL55) or phosphoprotein 65 (pp65) (UL83) were constructed and evaluated for their ability to induce immune responses in mice. The full-length gB as well as a truncated form expressing amino acids 1-680 of gB, and lacking the fragment encoding amino acids 681 907 including the transmembrane domain of gB (gB680) were evaluated. Immunization of mice with plasmids coding for gB or gB680 induced ELISA and neutralizing antibodies, with the highest titres in mice immunized with the gB680 plasmid. Mice immunized with the gB plasmid predominantly produced IgG2a gB-specific antibody, while the gB680 plasmid raised mostly IgG1 anti-gB antibody. Mice immunized with the pp65 plasmid developed pp65-specific cytotoxic T lymphocytes (CTL) and ELISA antibodies. Immunization with a mixture of both gB and pp65 plasmids raised antibodies to both proteins and pp65-specific CTL, indicating a lack of interference between these two plasmids. These results suggest that DNA immunization is a useful approach for vaccination against HCMV disease.     

Comparison of protective efficacies of plasmid DNAs encoding Japanese encephalitis virus proteins that induce neutralizing antibody or cytotoxic T lymphocytes in mice

Mice immunized with a plasmid DNA encoding the premembrane (prM) and envelope (E) proteins of Japanese encephalitis (JE) virus (designated pcJEME) produce neutralizing antibodies and are protected from JE. To determine the role of the immune response to other viral proteins in protection, we constructed plasmid DNAs encoding other JE virus proteins and made a direct comparison among these plasmids using a mouse model. Cytotoxic T lymphocytes (CTLs) were induced by plasmids encoding capsid (C) or nonstructural proteins, NS1, NS2A, NS2B, NS3 or NS5. However, these plasmids provided only a partial protection against intraperitoneal challenge with a lethal dose of JE virus, whereas mice immunized with pcJEME were fully protected. In mice inoculated with CTL-inducing plasmids, high virus titers were detected in plasma immediately (1h) following challenge and in brain on day 4 post-challenge, but no virus infectivity was detected in plasma and brain of pcJEME-immunized mice during the 5 days following challenge. These results indicate that protection provided by the prM/E-encoding DNA consists of neutralizing antibody that prevents virus dissemination from the peripheral site to the brain, and that this antibody-mediated mechanism of protection is more efficient than the immunity induced by plasmids that generate CTL responses capable of killing JE virus-infected cells.     

The cell-mediated immune response induced by plasmid encoding bovine herpesvirus 1 glycoprotein B is enhanced by plasmid encoding IL-12 when delivered intramuscularly or by gene gun, but not after intradermal injection

Bovine herpesvirus 1 (BHV-1) causes respiratory and genital infections in cattle. Previously we demonstrated that a DNA vaccine encoding a truncated, secreted form of BHV-1 glycoprotein B (tgB) induces cytotoxic T lymphocyte (CTL) responses in C3H mice. In this study we investigated the potential of interleukin 12 (IL-12) to further enhance the CTL response. C3H mice were immunized with a plasmid encoding tgB or with plasmids encoding tgB and murine IL-12. When the plasmid encoding tgB was delivered intramuscularly or epidermally by a gene gun, co-administration with IL-12 plasmid stimulated the synthesis of more IgG2a, the production of higher levels of IFN-gamma, and more effective killing by CTLs. In contrast, after intradermal delivery no effect of co-administration of IL-12 encoding plasmid was observed. Further investigation suggested that antigen and IL-12 need to be expressed in the draining lymph nodes, where IL-12 can have a direct effect on T cells.     

Vaccination of mice with DNA plasmids coding for the Chlamydia trachomatis major outer membrane protein elicits an immune response but fails to protect against a genital challenge

A DNA plasmid encoding the gene of the major outer membrane protein (MOMP) of the Chlamydia trachomatis mouse pneumonitis (MoPn) serovar and three plasmids containing the variable domains (VD) of the MOMP were constructed. Female mice were inoculated with the plasmids and 60 days later were challenged in the genital tract with C. trachomatis. Six weeks after challenge female mice were caged with male mice and the course of the mating followed. Mice immunized with the MOMP plasmids mounted weak humoral and cell mediated immune responses. However, following the genital challenge no significant differences in vaginal shedding were observed between the groups immunized with the MOMP and control plasmids. In addition, the fertility rates were similar in the experimental and negative control groups. In conclusion, vaccination with DNA plasmids encoding the MOMP elicited a modest immune response but did not protect against infection or disease.     

Immunization with plasmid DNA encoding the integral membrane protein, Sm23, elicits a protective immune response against schistosome infection in mice.

Schistosomes are helminth parasites infecting at least 200 million people worldwide. In this study, we evaluated the feasibility of using a nucleic acid vaccine to induce protective immune responses to the Schistosoma mansoni integral membrane protein Sm23. C57BL/6 mice were immunized by intramuscular injection in three separate vaccination trials. ELISA and Western Blot analyses indicated that mice immunized with a DNA plasmid construct encoding Sm23 (Sm23-pcDNA) generated specific IgG for Sm23, while sera from mice immunized with the control pcDNA plasmid did not. The vaccine elicited IgG(2a), and IgG(1) antibody isotypes. We also tested the adjuvant activity of IL-12 and IL-4 on humoral responses to Sm23. Co-immunization with plasmid encoding IL-12 did not affect the level of anti-Sm23 IgG(2a), but did reduce the IgG(1) level. In contrast, co-injection with a plasmid encoding IL-4 significantly reduced the level of anti-Sm23 IgG(2a), while the level of IgG(1) was largely unchanged. Importantly, the Sm23-pcDNA vaccine provided statistically significant levels of protection against challenge infection (21-44%, P<0.001-0.02). Co-administration of plasmids encoding either IL-12 or IL-4 did not significantly enhance this protective effect.     

Genetic vaccination against leishmaniasis

The gene encoding for a major surface glycoprotein, gp63, of Leishmania major was cloned into the eukaryotic expression plasmid pCDNAI with CMV or RSV promoters. The highly susceptible Balb/c mice were injected intramuscularly with 100 μg/mouse of the purified plasmid. The plasmids were found to be stable in vivo for at least 40 days after injection and expressed significant levels of gp63, demonstrable by immunohistological staining with specific antibody. The immunized mice developed significant resistance against L. major infection compared to controls similarly immunized with the empty plasmid. Spleen cells from the immunized mice produced significant levels of IL-2 and IFN-γ but no detectable IL-4 when cultured with leishmanial antigens in vitro.     

Vaccination with a human parainfluenza virus type 3 chimeric FHN glycoprotein formulated with a combination adjuvant induces protective immunity

Human parainfluenza virus type 3 (PIV3) is a major cause of lower respiratory disease i.e. bronchitis, bronchiolitis or pneumonia, in infants and young children. Presently there is no licensed vaccine against PIV3. To produce an effective subunit vaccine, a chimeric FHN glycoprotein consisting of the N-terminal ectodomain of the fusion (F) protein linked to the haemagglutinin-neuraminidase (HN) protein without transmembrane domain, and secreted forms of the individual F and HN glycoproteins, were expressed in mammalian cells and purified. Mice and cotton rats were immunized intramuscularly (IM) with FHN or both F and HN proteins (F + HN), formulated with poly(I:C) and an innate defense regulator peptide in polyphosphazene (TriAdj). Significantly higher levels of systemic virus-neutralizing antibodies were observed in mice and cotton rats immunized with FHN/TriAdj when compared to animals immunized with the combination of F and HN proteins (F + HN/TriAdj). As PIV3 is a pneumotropic virus, another goal is to produce an effective mucosal subunit vaccine. Intranasal (IN) administration with FHN/TriAdj resulted in mucosal IgA production in the lung and virus neutralizing antibodies in the sera. After PIV3 challenge no virus was detected in cotton rats immunized with FHN/TriAdj regardless of the route of delivery. Protective immunity against PIV3 was also induced by FHN/TriAdj in hamsters. In conclusion, the FHN protein formulated with TriAdj has potential for development of a safe and effective vaccine against PIV3.     

A novel immunization method to induce cytotoxic T-lymphocyte responses (CTL) against plasmid-encoded herpes simplex virus type-1 glycoprotein D

DNA molecules complexed with an asialoglycoprotein-polycation conjugate, consisting of asialoorosomucoid (ASOR) coupled to poly-L-lysine, can enter hepatocytes which bear receptors for ASOR. We used this receptor-mediated DNA delivery system to deliver plasmid DNA encoding glycoprotein D (gD) of herpes simplex virus type 1 to ASOR-positive cells. Maximum expression of gD protein was seen at 3 days after injection of this preparation in approximately 13% of cells from BALB/c mice [hepatocytes from mice injected intravenously (i.v.) or peritoneal exudate cells from mice injected intraperitoneally (i.p.)]. In comparison with mice injected with either the plasmid vector alone or the gD-containing plasmid uncomplexed to ASOR, mice immunized with gD-containing plasmid complexed with ASOR-poly-L-lysine induced marked antigen-specific CTL responses. BALB/c mice immunized with gD-DNA developed a T-cell-mediated CTL response against target cells expressing gD and MHC class II glycoproteins, but not against cells expressing only gD and MHC class I molecules. In C3H mice, gD-DNA induced a T-cell-mediated CTL response against target cells expressing gD and class I MHC molecules. Serum anti-gD antibody in low titers were produced in both strains of mice. DNA complexed with ASOR-poly-L-lysine induced CTL responses in mice.     

丙型肝炎病毒E2蛋白DNA疫苗诱导小鼠产生中和抗体的研究

目的探讨丙型肝炎病毒(HCV)包膜E2蛋白DNA疫苗诱导小鼠产生中和抗体的可行性。方法构建截除疏水性羧基末端的HCV包膜蛋白表达质粒pCI-1b661以及同时截除疏水性羧基末端和高变区1(HVR1)的表达质粒pCI-1b661Δ,转染293T细胞,以Western blot和ELISA检测细胞内和培养上清中的HCVE2蛋白,将两种表达质粒及空载体分别肌注免疫BALB/c小鼠,以ELISA检测小鼠血清中的HVR1抗体,以HCV假病毒颗粒(HCVpp)分析小鼠血清的中和活性。结果 2种表达质粒均能表达分泌性截短型E2蛋白。pCI-1b661免疫的8只小鼠血清中均可检测到HVR1抗体,而pCI-1b661Δ免疫血清中未检测到HVR1抗体。pCI-1b661和pCI-1b661Δ免疫血清对HCVpp的中和率分别为(78.5±13.8)%和(38.7±6.5)%,差异有显著性(P<0.01)。pCI-1b661免疫组小鼠血清的中和率与HVR1抗体水平呈正相关(r=0.967,P<0.01)。结论表达截短型E2蛋白的DNA疫苗能诱导产生HCV中和抗体,其主要成员为HVR1抗体。     

Immunological responses induced by a DNA vaccine expressing RON4 and by immunogenic recombinant protein RON4 failed to protect mice against chronic toxoplasmosis

The development of an effective vaccine against Toxoplasma gondii infection is an important issue due to the seriousness of the related public health problems, and the economic importance of this parasitic disease worldwide. Rhoptry neck proteins (RONs) are components of the moving junction macromolecular complex formed during invasion. The aim of this study was to evaluate the vaccine potential of RON4 using two vaccination strategies: DNA vaccination by the intramuscular route, and recombinant protein vaccination by the nasal route. We produced recombinant RON4 protein (RON4S2) using the Schneider insect cells expression system, and validated its antigenicity and immunogenicity. We also constructed optimized plasmids encoding full length RON4 (pRON4), or only the N-terminal (pNRON4), or the C-terminal part (pCRON4) of RON4. CBA/J mice immunized with pRON4, pNRON4 or pCRON4 plus a plasmid encoding the granulocyte-macrophage-colony-stimulating factor showed high IgG titers against rRON4S2. Mice immunized by the nasal route with rRON4S2 plus cholera toxin exhibited low levels of anti-RON4S2 IgG antibodies, and no intestinal IgA antibodies specific to RON4 were detected. Both DNA and protein vaccination generated a mixed Th1/Th2 response polarized towards the IgG1 antibody isotype. Both DNA and protein vaccination primed CD4+ T cells in vivo. In addition to the production of IFN-γ, and IL-2, Il-10 and IL-5 were also produced by the spleen cells of the immunized mice stimulated with RON4S2, suggesting that a mixed Th1/Th2 type immune response occurred in all the immunized groups. No cytokine was detectable in stimulated mesenteric lymph nodes from mice immunized by the nasal route. Immune responses were induced by both DNA and protein vaccination, but failed to protect the mice against a subsequent oral challenge with T. gondii cysts. In conclusion, strategies designed to enhance the immunogenicity and to redirect the cellular response towards a Th1 type response against RON4 could lead to more encouraging results.     

Priming with DNA plasmids encoding the nucleocapsid protein and glycoprotein precursors from Rift Valley fever virus accelerates the immune responses induced by an attenuated vaccine in sheep

In this work we tested the ability of plasmid DNA constructs encoding structural Rift Valley fever virus (RVFV) antigens to induce specific immune responses in sheep. The sole immunization of DNA constructs encoding the glycoprotein precursor NSm/G2/G1 did not suffice to induce a detectable antibody response. In contrast, immunization of sheep with a plasmid vector encoding the viral nucleocapsid protein N elicited a potent and long lasting induction of antibodies but with low neutralizing titers. After DNA immunization, no antigen-specific proliferating cells were detected in sheep PBLs. Boosting with the attenuated vaccine strain MP12 was able to increase the levels of proliferating memory cell pools and induction of IFN-gamma in response to purified virus or recombinant proteins, particularly in sheep vaccinated with a combination of both plasmid constructs. These results open the possibility to exploit this strategy to improve the induction of immune responses against RVFV in sheep.     

Immunity induced by DNA immunization with herpes simplex virus type 2 glycoproteins B and C

The complete sequence of herpes simplex virus type 2 (HSV-2) glycoproteins B and C (gB & gC) were cloned into plasmid expression vectors and evaluated in murine and guinea pig genital HSV-2 models. Balb/c mice were immunized with either pgB-2 or pgC-2 plasmids intramuscularly (IM) or intradermally (ID). The vaccines induced HSV-2-specific neutralizing and ELISA IgG antibody, but little or no enhancement of viral clearance from the vagina was detected following intravaginal challenge. Immunization of guinea pigs with pgB-2 or pgC-2 induced ELISA IgG antibody; however, antibody titers were approximately one log(10) unit lower than that seen in HSV-2 convalescent sera. IM immunization of guinea pigs with either plasmid also did not decrease vaginal viral shedding following vaginal challenge, but the severity of the acute disease and the subsequent number of recurrent lesion days were reduced in animals immunized with pgB-2. Lastly, IM immunization of latently infected guinea pigs with a combined gB-2 and gC-2 plasmid vaccine significantly reduced the number of subsequent HSV-2 recurrences. DNA vectors expressing gB-2 or gC-2 were both immunogenic, although the gB-2 plasmid induced higher titers of antibody and significantly reduced primary and recurrent herpetic disease in the guinea pig model. These results also suggest that immunotherapy with plasmid expression vectors may be effective against recurrent genital HSV-2 disease.     

Virus neutralizing antibody response in mice and dogs with a bicistronic DNA vaccine encoding rabies virus glycoprotein and canine parvovirus VP2

A bicistronic DNA vaccine against rabies and parvovirus infection of dogs was developed by subcloning rabies glycoprotein and canine parvovirus (CPV) VP2 genes into a bicistronic vector. After characterizing the expression of both the proteins in vitro, the bicistronic DNA vaccine was injected in mice and induced immune response was compared with monocistronic DNA vaccines. There was no significant difference in ELISA and virus neutralizing (VN) antibody responses against rabies and CPV in mice immunized with either bicistronic or monocistronic DNA vaccine. Further, there was significantly similar protection in mice immunized with either bicistronic or monocistronic rabies DNA vaccine on rabies virus challenge. Similarly, dogs immunized with monocistronic and bicistronic DNA vaccines developed comparable VN antibodies against rabies and CPV. This study indicated that bicistronic DNA vaccine can be used in dogs to induce virus neutralizing immune responses against both rabies and CPV.     

Immunization with plasmid DNA encoding a truncated, secreted form of the bovine viral diarrhea virus E2 protein elicits strong humoral and cellular immune responses

The major protective antigen of bovine viral diarrhea virus (BVDV), the E2 protein, is cell-associated and not expressed on the cell surface. In this study we evaluated a DNA vaccine encoding various secreted versions of E2. In vitro analysis demonstrated that deletion of the transmembrane anchor and addition of the signal sequence of bovine herpesvirus-1 (BHV-1) (gDsDeltaE2) resulted in efficient secretion of E2 into the culture medium. In contrast, full-length E2, either without or with gDs (gDsE2), as well as truncated E2 without gDs (DeltaE2), remained entirely cell-associated. Mice immunized with plasmid encoding gDsDeltaE2 developed significantly higher IgG and virus neutralizing antibody titres compared to animals vaccinated with plasmid encoding E2, DeltaE2 or gDsE2. To optimize secretion of E2, the efficiency of gDs was compared with that of the tissue plasminogen activator signal (tPAs) sequence. In addition, the effect of the plasmid backbone was assessed by comparing two vectors. Four plasmids, pMASIA-gDsDeltaE2, pMASIA-tPAsDeltaE2, pSLKIA-gDsDeltaE2 and pSLKIA-tPAsDeltaE2, were constructed and administered intradermally to mice. The mice immunized with pMASIA-tPAsDeltaE2 developed the strongest and most balanced immune responses. Vaccination of cattle confirmed that pMASIA-tPAsDeltaE2 elicited both strong humoral and cellular immune responses and thus could be a candidate DNA vaccine against BVDV.     

Immunization with plasmid DNA encoding the envelope glycoprotein of Japanese Encephalitis virus confers significant protection against intracerebral viral challenge without inducing detectable antiviral antibodies.

A plasmid DNA construct, pCMXENV encoding the envelope (E) glycoprotein of Japanese Encephalitis virus (JEV), was constructed. This plasmid expresses the E protein intracellularly, when transfected into Vero cells in culture. The ability of pCMXENV to protect mice from lethal JEV infection was evaluated using an intracerebral (i.c.) JEV challenge model. Several independent immunization and JEV challenge experiments were carried out and the results indicate that 51 and 59% of the mice are protected from lethal i.c. JEV challenge, when immunized with pCMXENV via intramuscular (i.m.) and intranasal (i.n.) routes respectively. None of the mice immunized with the vector DNA (pCMX) survived in any of these experiments. JEV-specific antibodies were not detected in pCMXENV-immunized mice either before or after challenge. JEV-specific T cells were observed in mice immunized with pCMXENV which increased significantly after JEV challenge indicating the presence of vaccination-induced memory T cells. Enhanced production of interferon-gamma (IFN-gamma) and complete absence of interleukin-4 (IL-4) in splenocytes of pCMXENV-immunized mice on restimulation with JEV antigens in vitro indicated that the protection is likely to be mediated by T helper (Th) lymphocytes of the Th1 sub-type. In conclusion, our results demonstrate that immunization with a plasmid DNA expressing an intracellular form of JEV E protein confers significant protection against i.c. JEV challenge even in the absence of detectable antiviral antibodies.     

Co-immunisation with a plasmid DNA cocktail primes mice against anthrax and plague

The protective antigen (PA) of Bacillus anthracis and the V antigen of Yersinia pestis are potent immunogens and candidate vaccine sub-units. When plasmid DNA encoding either PA or V antigen was used to immunise the Balb/c mouse, a low serum IgG titre was detected (log (10)1.0 or less) which was slightly increased by boosting with plasmid DNA. However, when mice immunised with plasmid DNA were later boosted with the respective recombinant protein, a significant increase in titre (up to 100-fold) was observed. Mice primed with a combination of each plasmid and boosted with a combination of the recombinant proteins, were fully protected (6/6) against challenge with Y. pestis. This compared favourably with mice primed only with plasmid DNA encoding the V antigen and boosted with rV, which were partially protected (3/6) against homologous challenge or with mice primed and boosted with plasmid DNA encoding the V antigen which were poorly protected (1/6). Combined immunisation with the two plasmid DNA constructs followed by boosting with a combination of the encoded recombinant proteins enhanced the protective immune response to Y. pestis compared with priming only with plasmid DNA encoding the V antigen and boosting with rV. This enhancement may be due to the effect of CpG motifs known to be present in the plasmid DNA construct encoding PA.     

Induced immune response of DNA vaccine encoding an association MSP1a, MSP1b, and MSP5 antigens of Anaplasma marginale

The outer membrane proteins of Anaplasma marginale have been the focus of research to obtain an improved vaccine against bovine anaplasmosis. We evaluated the capacity of the recombinant plasmids pcDNA-msp1alpha, pcDNA-msp1beta, and pcDNA-mp5 to express MSP1a, MSP1b, and MSP5 proteins, and to determine the immunogenicity of BALB/c mice immunized with these plasmids individually or in association. Expression of proteins was confirmed in Vero cells by IFA. The combination of recombinant plasmids showed high antibodies response, produced better induction of Th1 response than individual plasmids, and induced significant proliferation of splenocytes. The mice sera immunized with A. marginale showed seroconversion and reacted with all native MSPs, but demonstrated predominance of the humoral IgG1 isotype and did not induce significant proliferation of splenocytes. The use of association of recombinant plasmid can be an effective strategy for the immunoprophylaxis of anaplasmosis.     

A DNA vaccine encoding genetic fusions of carcinoembryonic antigen (CEA) and granulocyte/macrophage colony-stimulating factor (GM-CSF)

The anti-tumor immunologic effects of plasmid DNA vaccines encoding human carcinoembryonic antigen (CEA) fused to mouse granulocyte/macrophage colony-stimulating factor (GM-CSF) were examined. Immunization of C57BL/6 mice with the CEA-GMCSF fusion plasmids in a three injection, high-dose immunization schedule led to T cell and antibody responses specific for CEA. Mice injected with CEA-GMCSF fusion plasmids also developed IgG autoantibodies to GM-CSF. Tumor challenge with the CEA-expressing syngeneic mouse adenocarcinoma line, MC38-CEA-2, showed delayed tumor growth in mice immunized with the CEA-GMCSF fusion plasmids but complete protection in mice immunized with plasmid encoding CEA alone. In contrast, a single low-dose immunization with CEA-GMCSF fusion plasmids provided better tumor protection than low-dose CEA plasmid alone and resulted in lower titers of GM-CSF antibodies.     

Inactivated HSV-2 in MPL/alum adjuvant provides nearly complete protection against genital infection and shedding following long term challenge and rechallenge

Herpes Simplex Virus Type 2 (HSV-2) infection can result in life-long recurrent genital disease, asymptomatic virus shedding, and transmission. No vaccine to date has shown significant protection clinically. Here, we used a mouse model of genital HSV-2 infection to test the efficacy of a vaccine consisting of whole, formalin-inactivated HSV-2 (FI-HSV2) formulated with monophosphoryl lipid A (MPL) and alum adjuvants. Vaccine components were administered alone or as a prime-boost immunization together with DNA vaccines encoding a truncated glycoprotein D2 (gD2t) and two conserved HSV-2 genes necessary for virus replication, UL5 (DNA helicase) and UL30 (DNA polymerase). Our results show: (1) compared with mock immunized controls, mice immunized with FI-HSV2 plus MPL/alum consistently showed protection against disease burden and total viral shedding while the mice immunized with gD2t protein with MPL/alum did not; (2) protection against genital disease and viral replication correlated with the type of boost in a prime-boost immunization with little advantage afforded by a DNA prime; (3) intramuscular (i.m.) immunization with FI-HSV2 in MPL/Alhydrogel adjuvant provided nearly complete protection against vaginal HSV-2 shedding after a lethal intravaginal (i.vag.) short-term challenge and long-term rechallenge; (4) single formulation immunization with DNA vaccines, FI-HSV2, and MPL in an aluminum phosphate (Adju-Phos) adjuvant did not increase protection relative to FI-HSV2/MPL/Adju-Phos alone; and (5) addition of MPL/alum to the FI-HSV2 was required for optimal protection against disease, viral replication, and latent virus load in the dorsal root ganglia (DRG). Most notably, an optimized vaccine formulation of FI-HSV2 MPL/Alhydrogel given i.m. completely protected against detectable vaginal HSV-2 shedding in the majority of animals and HSV-2 latent DNA in the DRG of all animals.